Lin Amanda H Y, Sun Hui, Paudel Omkar, Lin Mo-Jun, Sham James S K
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
Cardiovasc Res. 2016 Jul 1;111(1):94-104. doi: 10.1093/cvr/cvw067. Epub 2016 Mar 24.
Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs).
PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T.
RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.
钙库操纵性钙内流（SOCE）在肺血管的多种生理和病理生理功能中发挥作用。由肌醇1,4,5 -三磷酸受体（InsP3R）门控钙库引起的SOCE已得到广泛研究，但兰尼碱受体（RyR）门控钙库在SOCE中的作用仍不清楚。本研究旨在阐明RyR门控钙库与SOCE之间的关系，并表征肺动脉平滑肌细胞（PASMCs）中RyR门控钙内流的特性。
从雄性Wister大鼠的叶内肺动脉分离出PASMCs。应用RyR1/2激动剂4 -氯间甲酚（4 - CmC）可激活PASMCs中强大的钙内流。它被钆（Gd³⁺）和RyR2调节剂K201阻断，但不受RyR1/3拮抗剂丹曲林和InsP3R抑制剂西司他汀C的影响，表明主要是RyR2参与了这一过程。小干扰RNA（siRNA）敲低基质相互作用分子1（STIM1）、瞬时受体电位通道蛋白1（TRPC1）和Orai1，或用ML - 9阻断STIM1易位，均显著减弱了4 - CmC诱导的SOCE，类似于毒胡萝卜素诱导的SOCE。然而，用咖啡因耗尽RyR门控钙库未能激活钙内流。加入本身不会引起钙内流的兰尼碱，以浓度依赖性方式揭示了咖啡因诱导的SOCE，表明兰尼碱与RyR的结合对这一过程是允许的。这种钙内流具有与4 - CmC诱导的SOCE相同的分子和药理学特性，并且一旦激活，即使在咖啡因洗脱后仍持续存在。对肌浆网（SR）中钙的测量表明，无论有无兰尼碱，应用4 - CmC和咖啡因都会使SR钙减少到相似程度，表明钙库耗竭不是差异的原因。此外，咖啡因/兰尼碱和4 - CmC未能在转染了兰尼碱结合缺陷突变体RyR2 - I4827T的细胞中引发SOCE。
RyR2门控钙库在PASMCs的SOCE中起作用；然而，仅钙库耗竭是不够的，还需要一种可被兰尼碱结合修饰的特定RyR构象来激活钙内流。
